Up until FY 2008, the ID09B time-resolved X-ray beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France was the only facility in the world capable of determining time-resolved macromolecular structures with 150-ps time resolution and <2-Angstrom spatial resolution. The Anfinrud group was instrumental in helping develop that capability at the ESRF. Unfortunately, the ESRF operated in a mode that was optimized for time-resolved Laue crystallography studies only 14 days out of each year, and we had access to only a portion of this limited amount of beam time. To expand the amount of beam time available for our studies, we partnered with the Advanced Photon Source (APS) in Argonne, IL and BioCARS to develop picosecond time-resolved X-ray capabilities on the ID14B beamline. In FY2005, Dr. Marvin Gershengorn, then Director of Intramural Research at NIDDK, committed >$1M to procure the capital equipment needed for this effort. Our vision was to achieve picosecond time-resolved X-ray capabilities comparable to that realized at the ESRF when the APS operates in 24-bunch mode, a common operating mode used 132 days per year. This goal required that we isolate a single bunch of X-rays from a train of pulses separated by only 153 ns, a feat that we first achieved in July 2007 using a high-speed chopper whose rotor was fabricated according to our custom specifications. To maximize the number of photons delivered to the sample in a single X-ray pulse, we replaced the existing U33 undulator (33-mm magnetic period) with two newly designed U23 and U27 undulators, making BioCARS the first APS beamline to operate with two inline undulators. NIDDK funded this effort, with the APS supplying the labor to design and refurbish two undulators according to our performance specifications. When the gaps of these undulators are tuned to generate 12-keV X-ray photons, the X-ray fluence is comparable to that generated at the ESRF during their 4-bunch mode. When the APS operates in their exotic hybrid mode, which is scheduled approximately 31 days per year, the X-ray fluence is a factor of 4 higher than that available with the ESRF 4-bunch mode. These achievements increased by more than an order of magnitude the amount of beamtime available worldwide to pursue 150 ps time-resolved X-ray science. The infrastructure needed to pursue picosecond time-resolved X-ray studies goes far beyond delivering single X-ray pulses to the experimental hutch. We also installed a $500K picosecond laser system in a laser hutch located near the X-ray hutch, as well as an array of laser diagnostics that aid optimization of the laser performance. We also developed a Field-Programmable-Gate-Array (FPGA) based timing system that synchronizes all time-critical components to the X-ray pulses. For example, the FPGA drives the heat-load chopper, the high-speed chopper, the picosecond laser system, a millisecond shutter, and various other motion controls that must be synchronized with the X-ray pulse arrival time. Importantly, we can set the time delay between X-ray and laser pulses from picoseconds to seconds with a precision of 10 ps. We also developed the diffractometer used to acquire time-resolved X-ray diffraction images. This effort included the design and fabrication of a millisecond shutter, a motorized support for the high-speed X-ray chopper, a support for motorized X-ray slits, detectors for non-invasively monitoring the laser and X-ray pulse energy and relative time delay, a motorized stage for the X-ray detector, supports for a collimator pipe and X-ray beam stop, beam conditioning optics that tailor the laser pulses in both space and time, beam delivery optics that focus the laser pulses onto the sample, motorized controls to center the focused laser pulse on the sample, and motorized controls to center the collimator pipe on the X-ray beam. Finally, we continue to refine the software developed to control the beamline. This software package, called LaueCollect, is written in the Python programming language, and is generalized for both time-resolved Laue crystallography and time-resolved SAXS/WAXS studies. In June 2013, we proposed a new BioCARS beamline layout that incorporates a secondary K-B mirror pair capable of focusing the X-ray beam to a spot size as small as 20 m. In January 2014, we received funding from NIDDK to acquire this mirror system. The K-B mirror is expected to arrive in Aug 2014, with X-ray commissioning and user operation scheduled for Oct 2014. The ability to focus the X-ray beam to much smaller dimensions will allow us and other BioCARS users to pursue time-resolved studies of small protein crystals. In March 2014 the BioCARS beam line acquired, with NIH support, a $1.6 million high-speed X-rays detector that will be a game changer in our time resolved X-ray studies. This detector is capable of acquiring high-resolution X-ray diffraction images at 10 Hz. In the past, the maximum rate at which we could write X-ray diffraction images to the file server was 0.25 Hz. Therefore, this new detector could speed our data collection 40 fold. To take advantage of the high-speed detector and the K-B mirror system, we have developed a next-generation high-speed diffractometer. This diffractometer incorporates a linear motor XYZ stage capable of 1g acceleration, and can move the sample to a new position in a few tens of milliseconds with 1-2 &#956;m precision. To facilitate the centering of the protein crystal on the rotation center of the phi motor, this diffractometer includes a pair of orthogonally-oriented color cameras: one provides one micron spatial resolution (10X objective), and the other offers a 6-mm wide field of view (1X objective). Moreover, this diffractometer is designed to accommodate both orthogonal and on-axis photo-excitation paths, and maintain a helium-purged path between the upstream components and the sample. The final assembly and testing this diffractometer is occurring in September 2014. The high-speed detector and high-speed diffractometer facilitates pair-wise acquisition of single-shot diffraction images, the first of which is acquired without laser excitation, and the second with laser education. Being single shot diffraction images, the signal-to-noise ratio achievable is limited. However, by acquiring many repeats of these image pairs and merging the results, we expect to achieve a level of signal-to-noise beyond what has been accomplished in the past. In addition to our instrument development efforts on the BioCARS beamline at the APS, we have developed the infrastructure required to pursue time-resolved X-ray studies at the Linear Coherent Light Source (LCLS) at Stanford with sub-ps time resolution. The LCLS is the worlds first free-electron X-ray laser, and is capable of generating extremely intense X-ray pulses with less than 100 fs duration. By exporting capabilities developed at the APS to the LCLS, we will extend the time resolution limits of our time-resolved X-ray studies by nearly 3 orders of magnitude. The improvements currently being made in both hardware and software pave the way to expand time-resolved Laue crystallography to many other protein systems, with our ultimate goal being the study of non-reversible enzymes on the chemical time scale of femtoseconds. The information gained from such studies will help unravel mysteries regarding how proteins function at a molecular level of detail.